During early embryogenesis, cells become determined to differentiate along specialized pathways. The molecular events underlying these processes are not understood. The long-term objective of this project is to obtain an understanding of the steps involved in nuclear determination and differentiation. To this end, special attention is being paid to changes in histone composition, histone modifications and chromatin structure in specific nuclei undergoing divergent pathways of differentiation. Conjugation in Tetrahymena will be used as a model to study nuclear determination and differentiation during development. In this system nuclei derived from daughter products of a single mitotic division differentiate into distinctly different products, macro- and micronuclei, depending upon their location within the cell. These end products are well characterized and large differences in structure and function exist between them. These differences must arise during conjugation. In many ways these events are directly analogous to cellular determination and differentiation which characterize early embryogenesis of higher animals. A variety of biochemical, cytological and immunological approaches will be used to 1) characterize the histone being synthesized and deposited in nuclei during various stages of conjugation and correlate any changes in histone synthesis with cytological stages; 2) isolate and fractionate different types of nuclei from mating cells in different stages; 3) define the histone composition and histone modifications associated with specific nuclei at different stages of differentiation; 4) probe the histone composition of specific nuclei in situ by immunofluorescent analyses and 5) isolate and characterize mononucleosomes containing minor histone variants from macronuclei of vegetative cells. Little is known about the mechanisms underlying nuclear (cellular) determination and differentiation, processes which characterize early development of higher eukaryotes. Understanding these mechanisms in normal development is essential if we are to understand cases of abnormal development, some congenital diseases, and certain pathological situations such as neoplastic transformation.